1. General Information
This specification contains amino acid sequence information prepared using Patent in Version 3.1, presented herein after the Abstract. Each sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length of each sequence and source organism are indicated by information provided in the numeric indicator fields <211> and <213>, respectively. Sequences referred to in the specification are defined by the term “SEQ ID NO:”, followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence designated as <400>1).
As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
All the references cited in this application are specifically incorporated by reference herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:    1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp1-22; Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;    4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;    5. Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text;    6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text;    7. Perbal, B., A Practical Guide to Molecular Cloning (1984);    8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;    9. J. F. Ramalho Ortigäo, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);    10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342    11. Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154.    12. Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.    13. Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.    14. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.    15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verilag, Heidelberg.    16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.    17. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).
2. Description of the Related Art
HCMV belongs to the θ-subfamily of the herpesvirus group, which are large strictly host-species specific DNA viruses encoding about 170-200 antigenically distinct polypeptides. HCMV is found universally throughout all geographic locations and socioeconomic groups, and infects between 50 and 85% of adults (Alford and Britt, In: Virology, 2nd Edition, Fields et. al. eds, Raven Press, 1990).
For most healthy persons who acquire primary HCMV infection after birth, there are few symptoms and no long-term health consequences. Occasionally, some adults with primary HCMV infection display symptoms of a mononucleosis-like syndrome with prolonged fever, and a mild hepatitis. Once infected with HCMV, the virus remains dormant by establishing a reservoir of latently-infected cells from which chronic low-grade re-activation into virus productive (lytic) cycle occurs throughout life. Although the factors controlling latency and re-activation are not completely understood, impairment of the body's cell-mediated immune system either by drug-induced immunosuppression or infection by certain pathogens can consistently reactivate the virus (Zaia and Forman, Infect Dis Clin North Am 9, 879-900, 1995).
There are clinical situations where HCMV infection is a significant cause of morbidity and mortality. For example, HCMV infection carries significant health risks to a foetus in utero, to people who work with children, and to individuals having a compromised immune system, such as, for example, those infected with HIV-1 or having undergone organ transplantation (Britt, Trends Microbiol 4, 34-81, 1996; Plotkin, Pediatr Infect Dis J 18, 313-325, 1999).
With particular respect to the health risks to a foetus in utero, those risks appear to be almost exclusively associated with non-immune women who become infected during pregnancy (Fowler et. al, New Engl J Med 326, 663-667, 1992; Murph et. al., In: Epidemiology of congenital cytomegalovirus infection: maternal risk factors and molecular analysis cytomegalovirus strains. 1998). Epidemiological studies have shown that 80%-90% of developing unborn babies who acquire congenital HCMV infection display a variable pattern of pathological sequelae within the first few years of life that may include hearing loss, vision impairment and mental retardation. Another 5% to 10% of infants who are infected but without symptoms at birth will subsequently have a varying degrees of hearing and mental or coordination problems. In 1996 alone, more than 17,000 cases of HCMV-induced sequelae or death were estimated in Europe and the USA (Plotkin, Pediatr Infect Dis J 18, 313-325, 1999).
Additionally, recent studies suggest that HCMV seropositive individuals who have undergone coronary angioplasty develop restenosis more frequently than seronegative patients (Field, Antivir Chem Chemother 10, 219-232, 1999), although a causal relationship has yet to be shown.
There is also a likelihood that there is a significant therapeutic benefit to be derived for individuals belonging to these high-risk groups, by reducing their HCMV load.
Accordingly, there is a need for an effective vaccine to provide such a reduction in HCMV load.
There have been a number of attempts at designing a vaccine against HCMV (for review see Britt, Trends Microbiol 4, 34-81, 1996; Plotkin, Pediatr Infect Dis J 18, 313-325, 1999), using either attenuated HCMV strains or subunit vaccines.
The first vaccines against HCMV were based on immunization using attenuated strains of HCMV, such as, for example, the Towne strain and AD-169 strain (Elek and Stern, Lancet 1, 1-5, 1974; Neff et. al., Proc Soc Exp Biol Med 160, 32-37, 1979). Although both attenuated viruses were shown to elicit cellular and humoral responses, neither vaccine prevented foetal infection in pregnant women experiencing a primary HCMV infection. Furthermore, vaccinated normal volunteers showed limited protection from viral challenge using the HCMV Toledo strain (Quinnan et. al., Ann Intern Med 101, 478-483, 1984; Adler et. al., Pediatr Infect Dis J. 17, 200-206, 1998). Subunit vaccines have been based on single HCMV antigen formulations, such as, for example, the full-length glycoprotein B (gB) polypeptide in combination with MF59 adjuvant (Chiron), or alternatively, a recombinant full-length gB polypeptide expressed using a viral vector (Pass et. al., J Infect Dis 180, 970-975, 1999; Adler et. al. J Infect Dis 180, 843-846, 1999). Additionally, a canarypox virus expressing a full length recombinant HCMV pp65 polypeptide has recently been tested in a clinical trial and shown to elicit a strong CTL and antibody response to this antigen (Gyulai et. al., Proceedings of the Seventh International Cytomegalovirus Workshop, Brighton, UK, Mar. 7-9, 1999, abstract).
However, vaccine formulations based on one or more full-length HCMV antigens are likely to present a number of limitations. For example, the expression of HCMV proteins such as pp65 can inhibit proteasomal processing of IE-1 through an associated kinase activity (Gilbert et al., Nature 383, 720, 1996). Moreover, other genes associated with the early phase of HCMV infection are also known to interfere at various steps of the MHC class I processing pathway and presentation (Reddehase, Curr. Opin. Immunol. 12, 390-396, 2000).
CTL epitope-based vaccines provide an alternative technology for overcoming the potential limitations associated with use of full-length HCMV antigens. However, the large degree of HLA polymorphism in human populations presents a major obstacle for the practical application of defined CTL epitopes as vaccines. A vaccine based on a single HCMV pp65-specific CTL epitope (NLVPMVATV; SEQ ID NO: 5) linked to the pan-HLA-DR T-helper epitope and one or two palmitic acid molecules is under investigation (Zaia et al., Hematol. (Am. Soc. Hematol. Educat. Program.) 339-355, 2000; La Rosa et al., Blood 97, 1776-1786, 2001).
For any subunit vaccine against HCMV, protection against HCMV is assumed to be achievable by inducing cellular immunity against a single virion antigen. There is evidence that in healthy carriers of the HCMV virus, subdominant T cell responses are also directed against other virion antigens, such as, for example, pp150, IE-1, and gH, which may also play a crucial role in controlling HCMV reactivation (for review see Britt, Trends Microbiol 4, 34-38, 1996; Ito, Nippon Rinsho 56, 62-68, 1998).
Clearly, the development of an effective vaccine against HCMV requires the elucidation of viral antigens that activate a protective cytotoxic T-lymphocyte (CTL) response and the determination of immunodominant CTL epitopes within those antigens.
CTL epitopes have been described for two immunodominant HCMV polypeptides, in particular, pp65 and IE-1 (Borysiewicz et al., J. Exp. Med. 168, 919, 1988; Sissons, J. Royal Coll. Phys., Lond. 20, 40, 1986; Wills et al., J. Virol. 70, 7569, 1996; Kern et al., Intervirology 42, 322, 1999; Weekes et al., J. Virol. 73, 2099, 1999; Reddehase, Curr. Opin. Immunol. 12, 390.-396, 2000).
Exemplary known CTL epitopes derived from HCMV pp65 are described herein With reference to SEQ ID NOs: 1-17 and 55. Epitopes listed as SEQ ID NOs: 1-17 are described as having the following HLA restrictions:    1. SVLGPISGHVLK (SEQ ID NO: 1) is restricted to HLA A*11xx (Diamond, U.S. Pat. No. 6,074,645, Jun. 13, 2000);    2. FTSQYRIQGKL (SEQ ID NO: 2) is restricted to HLA A*2402 (Longmate et al., Immunogenet. 52, 165-173, 2000);    3. FVFPTKDVALR (SEQ ID NO: 3) is restricted to HLA A*68xx (Longmate et al., Immunogenet. 52, 165-173, 2000);    4. FPTKDVAL (SEQ ID NO: 4) is restricted to HLA B35xx (Diamond U.S. Pat. No. 6,074,645, Jun. 13, 2000);    5. NLVPMVATV (SEQ ID NO: 5) is restricted to HLA A*02xx (Wills et al., J. Virol. 70, 7569-7579, 1996);    6. MLNIPSINV (SEQ ID NO: 6) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    7. RIFAELEGV (SEQ ID NO: 7) is restricted to HLA A*0201 (Diamond et al., Blood 90, 1751-1767, 1997);    8. TPRVTGGGGAM (SEQ ID NO: 8) is restricted to HLA B*07xx (Wills et al., J. Virol. 70, 7569-7579, 1996; Kern et al., Nature Med. 4, 975-978, 1998; Diamond U.S. Pat. No. 6,074,645, Jun. 13, 2000);    9. RPHERNGFTVL (SEQ ID NO: 9) is restricted to HLA B*07xx (Weekes et al., J. Virol. 73, 2099-2108, 1999; Diamond U.S. Pat. No. 6,074,645, Jun. 13, 2000);    10. RLLQTGIHV (SEQ ID NO: 10) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    11. VIGDQYVKV (SEQ ID NO: 11) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    12. ALFFFDIDL (SEQ ID NO: 12) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    13. YSEHPTFTSQY (SEQ ID NO: 13) is restricted to HLA A*01xx (Diamond, U.S. Pat. No. 6,074,645, Jun. 13, 2000);    14. VLCPKNMII (SEQ ID NO: 14) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    15. DIYRIFAEL (SEQ ID NO: 15) is restricted to HLA A*0201 (Solache et al., J. Immunol. 163, 5512-5518, 1999);    16. ILARNLVPMV (SEQ ID NO: 16) is restricted to HLA A*0201 (Diamond et al., Blood 90, 1751-1767, 1997; Solache et al., J. Immunol 163, 5512-5518, 1999); and    17. EFFWDANDIY (SEQ ID NO: 17) is restricted to HLA B*44xx (Longmate et al., Immunogenet. 52, 165-173, 2000).
The epitope listed in SEQ ID NO: 55 (IPSINVHHY) was described by Gavin et al., J. Immunol. 151, 3971-3980, 1993.
Exemplary known CTL epitopes derived from HCMV IE-1 are described herein with reference to SEQ ID NOs: 18-20. Respectively, those epitopes are described as having the following HLA restrictions:    1. YILEETSVM (SEQ ID NO: 18) is restricted to HLA A*02xx (Retière et al., J. Virol. 74, 3948-3952, 2000);    2. CVETMCNEY (SEQ ID NO: 19) is restricted to HLA B*18xx (Retière et al., J. Virol. 74, 3948-3952, 2000); and    3. RRIEEICMK (SEQ ID NO: 20) is restricted to HLA B*27xx (Salquin et al., Eur. J. Immunol. 30, 2531-2539, 2000).
Although there is some evidence to suggest that HCMV antigens other than pp65 or IE-1 may also be useful for eliciting CTL control of HCMV infection, such as, for example, HCMV pp150 and HCMV gB, information on the utility of those antigens is limited (Gyulai et al., J. Infect. Dis. 181,537-546, 2000). An exemplary known CTL epitope from HCMV pp150 is described herein with reference to SEQ ID NO: 21 having the following HLA restriction:    1. TTVYPPSSTAK (SEQ ID NO: 21) is restricted to HLA A*0301 (Longmate et al., Immunogenet. 52, 165-173, 2001).